Injectable composition containing crosslinkable material and cells for forming animal tissue

ABSTRACT

Slowly polymerizing polysaccharide hydrogels have been demonstrated to be useful as a means of delivering large numbers of isolated cells via injection. The gels promote engraftment and provide three dimensional templates for new cell growth. The resulting tissue is similar in composition and histology to naturally occurring tissue. This method can be used for a variety of reconstructive procedures, including custom molding of cell implants to reconstruct three dimensional tissue defects, as well as implantation of tissues generally.

This application is a continuation of U.S. patent application Ser. No.09/008,945, filed Jan. 20, 1998, now U.S. Pat. No. 6,730,298, which is acontinuation of U.S. patent application Ser. No. 08/056,140, filed Apr.30, 1993 and issued as U.S. Pat. No. 5,709,854, all of the above beingincorporated herein in their entirety by reference.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of creating new tissuesusing polysaccharide hydrogel-cell compositions.

Craniofacial contour deformities, whether traumatic, congenital, oraesthetic, currently require invasive surgical techniques forcorrection. Furthermore, deformities requiring augmentation oftennecessitate the use of alloplastic prostheses which suffer from problemsof infection and extrusion. A minimally invasive method of deliveringadditional autogenous cartilage or bone to the craniofacial skeletonwould minimize surgical trauma and eliminate the need for alloplasticprostheses. If one could transplant via injection and cause to engraftlarge numbers of isolated cells, one could augment the craniofacialosteo-cartilaginous skeleton with autogenous tissue, but withoutextensive surgery.

Unfortunately, attempts to inject dissociated cells subcutaneously or toimplant dissociated tissues within areas of the body such as theperitoneum have not been successful. Cells are relatively quicklyremoved, presumably by phagocytosis and cell death.

Cells can be implanted onto a polymeric matrix and implanted to form acartilaginous structure, as described in U.S. Pat. No. 5,041,138 toVacanti, et al., but this requires surgical implantation of the matrixand shaping of the matrix prior to implantation to form a desiredanatomical structure.

Accordingly, it is an object of the present invention to provide amethod and compositions for injection of cells to form cellular tissuesand cartilaginous structures.

It is a further object of the invention to provide compositions to formcellular tissues and cartilaginous structures including non-cellularmaterial which will degrade and be removed to leave tissue or cartilagethat is histologically and chemically the same as naturally producedtissue or cartilage.

Summary of the Invention

Slowly polymerizing, biocompatible, biodegradable hydrogels have beendemonstrated to be useful as a means of delivering large numbers ofisolated cells into a patient to create an organ equivalent or tissuesuch as cartilage. The gels promote engraftment and provide threedimensional templates for new cell growth. The resulting tissue issimilar in composition and histology to naturally occurring tissue. Inone embodiment, cells are suspended in a hydrogel solution and injecteddirectly into a site in a patient, where the hydrogel hardens into amatrix having cells dispersed therein. In a second embodiment, cells aresuspended in a hydrogel solution which is poured or injected into a moldhaving a desired anatomical shape, then hardened to form a matrix havingcells dispersed therein which can be be implanted into a patient.Ultimately, the hydrogel degrades, leaving only the resulting tissue.

This method can be used for a variety of reconstructive procedures,including custom molding of cell implants to reconstruct threedimensional tissue defects, as well as implantation of tissuesgenerally.

DETAILED DESCRIPTION OF THE INVENTION

Techniques of tissue engineering employing biocompatible polymerscaffolds hold promise as a means of creating alternatives to prostheticmaterials currently used in craniomaxillofacial surgery, as well asformation of organ equivalents to replaced diseased, defective, orinjured tissues. However, polymers used to create these scaffolds, suchas polylactic acid, polyorthoesters, and polyanhydrides, are difficultto mold and hydrophobic, resulting in poor cell attachment. Moreover,all manipulations of the polymers must be performed prior toimplantation of the polymeric material.

Calcium alginate and certain other polymers can form ionic hydrogelswhich are malleable and can be used to encapsulate cells. In thepreferred embodiment described herein, the hydrogel is produced bycross-linking the anionic salt of alginic acid, a carbohydrate polymerisolated from seaweed, with calcium cations, whose strength increaseswith either increasing concentrations of calcium ions or alginate. Thealginate solution is mixed with the cells to be implanted to form analginate suspension. Then, in one embodiment, the suspension is injecteddirectly into a patient prior to hardening of the suspension. Thesuspension then hardens over a short period of time. In a secondembodiment, the suspension is injected or poured into a mold, where ithardens to form a desired anatomical shape having cells dispersedtherein.

Polymeric Materials.

The polymeric material which is mixed with cells for implantation intothe body should form a hydrogel. A hydrogel is defined as a substanceformed when an organic polymer (natural or synthetic) is cross-linkedvia covalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate, polyphosphazines, and polyacrylates,which are crosslinked ionically, or block copolymers such as Pluronics™or Tetronics™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups.

Examples of polymers with basic side groups that can be reacted withanions are poly(vinyl amines), poly(vinyl pyridine), poly(vinylimidazole), and some imino substituted polyphosphazenes. The ammonium orquaternary salt of the polymers can also be formed from the backbonenitrogens or pendant imino groups. Examples of basic side groups areamino and imino groups.

Alginate can be ionically cross-linked with divalent cations, in water,at room temperature, to form a hydrogel matrix. Due to these mildconditions, alginate has been the most commonly used polymer forhybridoma cell encapsulation, as described, for example, in U.S. Pat.No. 4,352,883 to Lim. In the Lim process, an aqueous solution containingthe biological materials to be encapsulated is suspended in a solutionof a water soluble polymer, the suspension is formed into droplets whichare configured into discrete microcapsules by contact with multivalentcations, then the surface of the microcapsules is crosslinked withpolyamino acids to form a semipermeable membrane around the encapsulatedmaterials.

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains (“R”). Therepeat unit in polyphosphazenes has the general structure (1):

-   -   where n is an integer.

The polyphosphazenes suitable for cross-linking have a majority of sidechain groups which are acidic and capable of forming salt bridges withdi- or trivalent cations. Examples of preferred acidic side groups arecarboxylic acid groups and sulfonic acid groups. Hydrolytically stablepolyphosphazenes are formed of monomers having carboxylic acid sidegroups that are crosslinked by divalent or trivalent cations such asCa²⁺ or Al³⁺. Polymers can be synthesized that degrade by hydrolysis byincorporating monomers having imidazole, amino acid ester, or glycerolside groups. For example, a polyanionicpoly[bis(carboxylatophenoxy)]phosphazene (PCPP) can be synthesized,which is cross-linked with dissolved multivalent cations in aqueousmedia at room temperature or below to form hydrogel matrices.

Bioerodible polyphosphazines have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol andglucosyl. The term bioerodible or biodegrable, as used herein, means apolymer that dissolves or degrades within a period that is acceptable inthe desired application (usually in vivo therapy), less than about fiveyears and most preferably less than about one year, once exposed to aphysiological solution of pH 6-8 having a temperature of between about25° C. and 38° C. Hydrolysis of the side chain results in erosion of thepolymer. Examples of hydrolyzing side chains are unsubstituted andsubstituted imidizoles and amino acid esters in which the group isbonded to the phosphorous atom through an amino linkage (polyphosphazenepolymers in which both R groups are attached in this manner are known aspolyaminophosphazenes). For polyimidazolephosphazenes, some of the “R”groups on the polyphosphazene backbone are imidazole rings, attached tophosphorous in the backbone through a ring nitrogen atom. Other “R”groups can be organic residues that do not participate in hydrolysis,such as methyl phenoxy groups or other groups shown in the scientificpaper of Allcock, et al., Macromolecule 10:824-830 (1977).

Methods for synthesis and the analysis of various types ofpolyphosphazenes are described by Allcock, H. R.; et al., Inorg. Chem.11, 2584 (1972); Allcock, et al., Macromolecules 16, 715 (1983);Allcock, et al., Macromolecules 19, 1508 (1986); Allcock, et al.,Biomaterials, 19, 500 (1988); Allcock, et al., Macromolecules 21, 1980(1988); Allcock, et al., Inorg. Chem. 21(2), 515-521 (1982); Allcock, etal., Macromolecules 22, 75 (1989); U.S. Pat. Nos. 4,440,921, 4,495,174and 4,880,622 to Allcock, et al.; U.S. Pat. No. 4,946,938 to Magill, etal.; and Grolleman, et al., J. Controlled Release 3, 143 (1986), theteachings of which are specifically incorporated herein by reference.

Methods for the synthesis of the other polymers described above areknown to those skilled in the art. See, for example Concise Encyclopediaof Polymer Science and Polymeric Amines and Ammonium Salts, E. Goethals,editor (Pergamen Press, Elmsford, N.Y. 1980). Many polymers, such aspoly(acrylic acid), are commercially available.

The water soluble polymer with charged side groups is crosslinked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups or multivalent anions if the polymer has basicside groups. The preferred cations for cross-linking of the polymerswith acidic side groups to form a hydrogel are divalent and trivalentcations such as copper, calcium, aluminum, magnesium, strontium, barium,and tin, although di-, tri- or tetra-functional organic cations such asalkylammonium salts, e.g., R₃N⁺—\ /\ /\ /—⁺NR₃ can also be used. Aqueoussolutions of the salts of these cations are added to the polymers toform soft, highly swollen hydrogels and membranes. The higher theconcentration of cation, or the higher the valence, the greater thedegree of cross-linking of the polymer. Concentrations from as low as0.005 M have been demonstrated to cross-link the polymer. Higherconcentrations are limited by the solubility of the salt.

The preferred anions for cross-linking of the polymers to form ahydrogel are divalent and trivalent anions such as low molecular weightdicarboxylic acids, for example, terepthalic acid, sulfate ions andcarbonate ions. Aqueous solutions of the salts of these anions are addedto the polymers to form soft, highly swollen hydrogels and membranes, asdescribed with respect to cations.

A variety of polycations can be used to complex and thereby stabilizethe polymer hydrogel into a semi-permeable surface membrane. Examples ofmaterials that can be used include polymers having basic reactive groupssuch as amine or imine groups, having a preferred molecular weightbetween 3,000 and 100,000, such as polyethylenimine and polylysine.These are commercially available. One polycation is poly(L-lysine);examples of synthetic polyamines are: polyethyleneimine,poly(vinylamine), and poly(allyl amine). There are also naturalpolycations such as the polysaccharide, chitosan.

Polyanions that can be used to form a semi-permeable membrane byreaction with basic surface groups on the polymer hydrogel includepolymers and copolymers of acrylic acid, methacrylic acid, and otherderivatives of acrylic acid, polymers with pendant SO₃H groups such assulfonated polystyrene, and polystyrene with carboxylic acid groups.

Sources of Cells.

Cells can be obtained directed from a donor, from cell culture of cellsfrom a donor, or from established cell culture lines. In the preferredembodiments, cells are obtained directly from a donor, washed andimplanted directly in combination with the polymeric material. The cellsare cultured using techniques known to those skilled in the art oftissue culture.

Cell attachment and viability can be assessed using scanning electronmicroscopy, histology, and quantitative assessment with radioisotopes.The function of the implanted cells can be determined using acombination of the above-techniques and functional assays. For example,in the case of hepatocytes, in vivo liver function studies can beperformed by placing a cannula into the recipient's common bile duct.Bile can then be collected in increments. Bile pigments can be analyzedby high pressure liquid chromatography looking for underivatizedtetrapyrroles or by thin layer chromatography after being converted toazodipyrroles by reaction with diazotized azodipyrrolesethylanthranilate either with or without treatment with P-glucuronidase.Diconjugated and monoconjugated bilirubin can also be determined by thinlayer chromatography after alkalinemethanolysis of conjugated bilepigments. In general, as the number of functioning transplantedhepatocytes increases, the levels of conjugated bilirubin will increase.Simple liver function tests can also be done on blood samples, such asalbumin production. Analogous organ function studies can be conductedusing techniques known to those skilled in the art, as required todetermine the extent of cell function after implantation. For example,islet cells of the pancreas may be delivered in a similar fashion tothat specifically used to implant hepatocytes, to achieve glucoseregulation by appropriate secretion of insulin to cure diabetes. Otherendocrine tissues can also be implanted. Studies using labelled glucoseas well as studies using protein assays can be performed to quantitatecell mass on the polymer scaffolds. These studies of cell mass can thenbe correlated with cell functional studies to determine what theappropriate cell mass is. In the case of chondrocytes, function isdefined as providing appropriate structural support for the surroundingattached tissues.

This technique can be used to provide multiple cell types, includinggenetically altered cells, within a three-dimensional scaffolding forthe efficient transfer of large number of cells and the promotion oftransplant engraftment for the purpose of creating a new tissue ortissue equivalent. It can also be used for immunoprotection of celltransplants while a new tissue or tissue equivalent is growing byexcluding the host immune system.

Examples of cells which can be implanted as described herein includechondrocytes and other cells that form cartilage, osteoblasts and othercells that form bone, muscle cells, fibroblasts, and organ cells. Asused herein, “organ cells” includes hepatocytes, islet cells, cells ofintestinal origin, cells derived from the kidney, and other cells actingprimarily to synthesize and secret, or to metabolize materials.

Addition of Biologically Active Materials to the Hydrogel.

The polymeric matrix can be combined with humoral factors to promotecell transplantation and engraftment. For example, the polymeric matrixcan be combined with angiogenic factors, antibiotics,antiinflammatories, growth factors, compounds which inducedifferentiation, and other factors which are known to those skilled inthe art of cell culture.

For example, humoral factors could be mixed in a slow-release form withthe cell-alginate suspension prior to formation of implant ortransplantation. Alternatively, the hydrogel could be modified to bindhumoral factors or signal recognition sequences prior to combinationwith isolated cell suspension.

Methods of Implantation.

The techniques described herein can be used for delivery of manydifferent cell types to achieve different tissue structures. In thepreferred embodiment, the cells are mixed with the hydrogel solution andinjected directly into a site where it is desired to implant the cells,prior to hardening of the hydrogel. However, the matrix may also bemolded and implanted in one or more different areas of the body to suita particular application. This application is particlularly relevantwhere a specific structural design is desired or where the area intowhich the cells are to be implanted lacks specific structure or supportto facilitate growth and proliferation of the cells.

The site, or sites, where cells are to be implanted is determined basedon individual need, as is the requisite number of cells. For cellshaving organ function, for example, hepatocytes or islet cells, themixture can be injected into the mesentery, subcutaneous tissue,retroperitoneum, properitoneal space, and intramuscular space. Forformation of cartilage, the cells are injected into the site wherecartilage formation is desired. One could also apply an external mold toshape the injected solution. Additionally, by controlling the rate ofpolymerization, it is possible to mold the cell-hydrogel injectedimplant like one would mold clay.

Alternatively, the mixture can be injected into a mold, the hydrogelallowed to harden, then the material implanted.

Specific Applications.

This technology can be used for a variety of purposes. For example,custom-molded cell implants can be used to reconstruct three dimensionaltissue defects, e.g., molds of human ears could be created and achondrocyte-hydrogel replica could be fashioned and implanted toreconstruct a missing ear. Cells can also be transplanted in the form ofa thee-dimensional structure which could be delivered via injection.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Preparation of a Calcium-Alginate-chondrocyte Mixture andInjection into Mice to Form Cartilaginous Structures.

A calcium alginate mixture was obtained by combining calcium sulfate, apoorly soluble calcium salt, with a 1% sodium alginate dissolved in a0.1 M potassium phosphate buffer solution (pH 7.4). The mixture remainedin a liquid state at 4° C. for 30-45 min. Chondrocytes isolated from thearticular surface of calf forelimbs were added to the mixture togenerate a final cellular density of 1×10⁷/ml (representingapproximately 10% of the cellular density of human juvenile articularcartilage).

The calcium alginate-chondrocyte mixture was injected through a 22 gaugeneedle in 100 μl aliquots under the pannus cuniculus on the dorsum ofnude mice.

The nude mice were examined 24 hours postoperatively, and all injectionsites were firm to palpation without apparent diffusion of the mixture.Specimens were harvested after 12 weeks of in vivo incubation. On grossexamination, the calcium alginate-chondrocyte specimens exhibited apearly opalescence and were firm to palpation. The specimens weighed0.11±0.01 gms (initial weight 0.10 gms). The specimens were easilydissected free of surrounding tissue and exhibited minimal inflammatoryreaction. Histologically, the specimens were stained with hematoxylinand eosin and demonstrated lacunae within a basophilic ground glasssubstance.

Control specimens of calcium alginate without chondrocytes had a doughyconsistency 12 weeks after injection and had no histologic evidence ofcartilage formation.

This study demonstrates that an injectable calcium alginate matrix canprovide a three dimensional scaffold for the successful transplantationand engraftment of chondrocytes. Chondrocytes transplanted in thismanner form a volume of cartilage after 12 weeks of in vivo incubationsimilar to that initially injected.

EXAMPLE 2 Effect of Cell Density on Cartilage Formation.

Varying numbers of chondrocytes isolated from the articular surface ofcalf forelimbs were mixed with a 1.5% sodium alginate solution togenerate final cell densities of 0.0, 0.5, 1.0, and 5.0×10⁶chondrocytes/ml (approximately 0.0, 0.5, 1.0, and 5.0% of the cellulardensity of human juvenile articular cartilage). An aliquot of thechondrocyte-alginate solution was transferred to a circular mold 9 mm indiameter and allowed to polymerize at room temperature by the diffusionof a calcium chloride solution through a semi-permeable membrane at thebase of the mold. The gels formed discs measuring 2 mm in height and 9mm in diameter.

Discs of a fixed cellular density of 5×10⁶ cells/ml were also formed inwhich the concentration of the sodium alginate and the molarity of thecalcium chloride solutions were varied.

All discs were placed into dorsal subcutaneous pockets in nude mice.Samples were harvested at 8 and 12 weeks and examined for gross andhistological evidence of cartilage formation.

Examinations of 8 and 12 week specimens revealed that a minimum celldensity of 5×10⁶ chondrocytes/ml was required for cartilage productionwhich was observed only 12 weeks after implantation on grossexamination, the specimens were discoid in shape and weighed 0.13±0.01gms (initial weight 0.125 gms). The specimens were easily dissected freeof surrounding tissue and exhibited minimal inflammatory reaction.Histologically, the specimens were stained with hematoxylin and eosinand demonstrated lacunae within a basophilic ground glass substance.

Cartilage formation was independent of calcium chloride concentrationused in gel polymerization. Cartilage was observed in specimens withalginate concentrations varying from 0.5% to 4.0%; however, the lowestalginate concentration tested (0.5%) showed only microscopic evidence ofcartilage.

Cartilage can be grown in a subcutaneous pocket to a pre-determined discshape using calcium alginate gel as a support matrix in 12 weeks.Cartilage formation is not inhibited by either polymerization with highcalcium concentrations or the presence of high alginate concentrationsbut does require a minimum cellular density of 5×10⁶ cells/ml.

The ability to create a calcium alginate-chondrocyte gel in a givenshape demonstrates that it is possible to use this technique to customdesign and grow cartilaginous scaffolds for craniofacial reconstruction.Such scaffolds have the potential to replace many of the prostheticdevices currently in use.

EXAMPLE 3 Preparation of Implantable Premolded Cell-polymer Mixtures.

250 μl aliquots of an isolated chondrocyte suspension was mixed with 750μls of a 2% (w/v) sodium alginate solution (0.1 M K₂HPO₄, 0.135 M NaCl,pH 7.4). A 125 μl aliquot was placed into 9 mm diameter cell cultureinserts with 0.45 μm pore size semipermeable membranes. Thecell-alginate mixture was placed into contact with a 30 mM CaCl₂ bathand allowed to polymerize for 90 minutes at 37° C. After 90 minutes, thecell-alginate gel constructs were removed from the mold and had adiameter of 9 mm and a height of 2 mm. The discs were placed into thewells of 24-well tissue culture plates and incubated at 37° C. in thepresence of 5% CO₂ with 0.5 ml of a solution containing Hamm's F-12culture media (Gibco, Grand Island, N.Y.) and 10% fetal calf serum(Gibco, Grand Island, N.Y.) with L-glutamine (292 μg/ml), penicillin(100 U/ml), streptomycin (100 μg/ml) and ascorbic acid (5 μg/ml) for 48hrs.

Using this method, bovine chondrocyte-alginate discs were prepared, thenimplanted in dorsal subcutaneous pockets in athymic mice using standardsterile technique. After one, two, and three months, athymic mice weresacrificed, and the gel/chondrocyte constructs removed, weighed andplaced in appropriate fixative. The cell-polymer complexes were studiedby histochemical analysis.

Cartilage formation was observed histologically after three months of invivo incubation at an initial chondrocyte density of 5×10⁶ cell/ml.

The above protocol was modified by using a range of CaCl₂ concentrationand a range of sodium alginate concentrations. Cartilage formation wasobserved using 15, 20, 30, and 100 mM CaCl₂ baths and 0.5, 1.0, 1.5,2.0, and 4.0% sodium alginate solutions.

By changing the mold within which the cell-alginate construct iscreated, the shape of the implant can be customized. Additionally, themold need not be semipermeable as calcium ion can be directly mixed withthe cell-alginate solution prior to being placed within a mold. The keyfeature is that the construct can be fashioned into a given shape priorto implantation.

EXAMPLE 4 Preparation of Injectable Osteoblasts-hydrogel Mixtures.

Using the methodology described above, bovine osteoblasts have beensubstituted for chondrocytes and injected into animals using a hydrogelmatrix.

Histology after 12 weeks of in vivo incubation showed the presence ofearly bone formation.

EXAMPLE 5 Use of the hydrogel matrix to form an immunoprotective matrixaround the implanted cells.

By fashioning a cell-alginate construct as described above, one can usethe hydrogel matrix to sterically isolate the encapsulated cells fromthe host immune system, and thereby allow allogenic cell transplants toform new tissues or organs without immunosuppression.

Bovine chondrocytes in an alginate suspension were transplanted intonormal immune-competent mice. Histology after six weeks of in vivoincubation shows the presence of cartilage formation. Gross examinationof the specimens does not demonstrate features of cartilage. Literaturestates that similar chondrocyte xenografts without alginate do not formcartilage.

Modifications and variations of the compositions and methods of thepresent invention will be obvious to those skilled in the art from theforegoing detailed description. Such modifications and variations areintended to come within the scope of the following claims.

1. A composition for forming tissue in an animal comprising (a) aninjectable solution of an ionically crosslinkable material forming ahydrogel when crosslinked, (b) an agent capable of ionicallycrosslinking the material, and (c) dissociated cells selected from thegroup consisting of cells that form cartilage, muscle cells,fibroblasts, and organ cells, wherein the cells are suspended in thecrosslinkable material and the crosslinking agent is mixed with the cellsuspension to form a hydrogel having cells suspended therein.
 2. Thecomposition of claim 1 wherein the material capable of forming ahydrogel is selected from the group consisting of alginate,polyphosphazincs, polyethylene oxide-polypropylene glycol blockcopolymers, poly(acrylic acids), poly(methacrylic acids), copolymers ofacrylic acid and methacrylic acid, poly(vinyl acetate), and sulfonatedpolymers.
 3. The composition of claim 2 wherein the ionicallycrosslinked material is further stabilized by cross-linking with apolyion.
 4. The composition of claim 1 wherein the cells are organcells.
 5. The composition of claim 1 wherein the cells are chondrocytes.6. The composition of claim 1 wherein between 1 and 5 million cells aresuspended per ml of the composition.
 7. The composition of claim 1wherein between 0.5 and 5% of the cellular density of human juvenilearticular cartilage cells are suspended per ml of the composition. 8.The composition of claim 1 wherein crosslinking of the hydrogel solutionis initiated prior to implantation in the animal.
 9. The composition ofclaim 1 wherein the hydrogel is injected into the animal as a cellsuspension, which then crosslinks to form a hydrogel-cell suspension.10. The composition of claim 2 wherein the crosslinking agent iscalcium.
 11. The composition of claim 1 wherein the agent capable ofcrosslinking the material comprises cations or anions, wherein thecations are selected from the group consisting of copper, calcium,aluminum, magnesium, strontium, barium, tin, and di-, tri- ortetra-functional organic cations; and the anions are selected from thegroup consisting of low molecular weight dicarboxylic acids, sulfateions and carbonate ions.